College of Agriculture Administration

Area of Expertise: Structural Basis for RNA function
RNA is not a passive messenger
In the post genomic age, non-coding RNA sequences
(those that do not encode proteins) have been found unexpectedly
to be very important for regulation of gene expression and other
cellular processes. Many of these are functional RNAs that can
fold into stable three-dimensional structures and thereby
perform work in the cell. A wide variety of riboswitches, which
toggle gene expression on and off, have been identified, and
catalytic RNAs, or ribozymes, are being discovered in a variety
of contexts, including eukaryotic transcriptomes. It is
therefore essential to develop a detailed understanding of these
systems in order to understand the catalytic and regulatory
functions of ribozymes and riboswitches. In
our laboratory, we undertake structural and mechanistic analyses
of functional RNAs and ribozymes to provide a picture of how
these molecules can work within the cell.

The HDV ribozyme has a hybrid engine
The hepatitis delta virus (HDV) ribozyme is a
small ribozyme originally identified in the genome of the HDV.
HDV is a human pathogen that co-infects with the hepatitis B
virus and thereby leads to liver disease. The ribozyme
self-cleaves viral RNAs, synthesized as tandem repeats during
rolling circle replication, into genome-sized pieces. This
ribozyme has evolved to function within human cells and
therefore has the potential to be used as a therapeutic and can
be used as a molecular biological tool. Recently, functional
HDV-like ribozymes have been identified within the human
transcriptome, in plants, fungi and insects, and in the mosquitoAnopheles
gambiaewhere
cleavage is activated in a developmentally-regulated fashion. Ribozyme self-cleavage thus represents a potential target for
mosquito-borne pathogens, including Dengue virus, West Nile
virus, and Malaria.

To create a snapshot of the ribozyme, we have
trapped the molecule in a state prior to cleavage and solved its
crystal structure. This gives us a picture of an RNA molecule
poised to react. At the cleavage site, we observe an RNA
nucleobase, cytosine 75 (C75), and a magnesium ion interacting
with the cleavage site. This structure confirms a reaction
mechanism in which C75 is initially protonated. We have shown
that C75 possesses a dramatically shifted pK and
can therefore participate in proton transfer reactions at
neutral pH. This property allows it to donate the proton to the
5’-hydroxyl leaving group, thereby activating it for catalysis.
Surprisingly, we saw a magnesium ion interacting with both the
2’-hydroxyl attacking group and the cleavage site phosphate to
help position the substrate for cleavage and to activate the
nucleophile. In the use of a magnesium ion in the cleavage
reaction, the HDV ribozyme functions similarly to the group I
introns. This mechanism represents a paradigm shift because the
ribozymes, such as the HDV ribozyme, were thought to be too
small to bind and position metals for Lewis acid catalysis, a
mechanism in which the metal ion interacts directly with the
2’-hydroxyl nucleophile. By both positioning a metal ion and by
shifting the pKof
C75, the HDV ribozyme overcomes the intrinsic inertness of RNA.
Thus, the HDV ribozyme is the first ribozyme that has been
observed to use two distinct catalytic strategies to perform an
RNA cleavage reaction.

These studies have provided new concepts in the
understanding of RNA structure and RNA catalytic strategies that
will aid in the understanding of other RNA catalysts. It is
likely that the genomic sequencing enterprises will reveal new
RNA catalysts that will either be widespread across evolution,
or unique to individual organisms. Our work will aid in the
process of identifying and characterizing novel ribozymes.

Awards & Honors

(2014) Co-Organizer. 19th Annual Meeting of the RNA Society.

(2014) Member. NIH MSFB Study Section.

(2014) University Faculty Scholar. Purdue University.

(2013) Member. NIH MSFB Study Section.

(2012) Lead Organizer. FASEB Nucleic Acid Enzymes Conference.

(2012) Member. NIH MSFB Study Section.

(2011) Seed for Success Award. Purdue University.

(2000) PEW Scholar. The Pew Scholars Program in the Biomedical Sciences.